This invention relates to an acridinium compound which may be used as a chemiluminescent label in a specific binding assay, and a conjugate thereof, and more specifically, to an acridinium compound which exhibits a high chemiluminescence yield enabling a high sensitive specific binding assay, and a conjugate thereof.
Acridinium compounds are useful for a chemiluminescent label since their luminescence efficiency is higher than other chemiluminescent compounds such as luminol. A chemiluminescent label is not only required to have a high luminescence efficiency but also required to have a functional group that enables the labeling procedure, and a stability of practical level upon use in the specific binding assay whether the compound is used as it is or after forming a conjugate with the specific binding substance.
An acridinium compound used for such chemiluminescent label should contain a functional group which has a covalent bonding activity with the so called specific binding substance so that the functional group may serve a binding group upon binding with the specific binding substance. A specific binding substance is a substance that constitutes a specific binding pair such as an antigen and an antibody and other immunological pairs; a lectin and a sugar chain; a ligand and a receptor; an enzyme and a substrate; and a target nucleic acid and its complementary nucleic acid. The acridinium compound used for the chemiluminescent label should also contain a moiety which serve a leaving group upon formation of the dioxetane on the carbon atom at 9-position in the acridinium ring. The moiety of the leaving group is typically phenoxy group which is bonded to the carbon atom at 9-position of the acridinium ring with the intervening carbonyl group. There are two types of compounds which differ in the location of the functional group for the labeling purpose. In one type of the compounds, the functional group for the labeling purpose is located on the part of the phenoxy group which is bonded to the carbon atom at 9-position of the acridinium ring with the intervening carbonyl group. In another type of the compounds, the functional group for the labeling purpose is located on the part of the nitrogen atom at 10-position of the acridinium ring.
The compound having the functional group for the labeling purpose on the part of the leaving group is significantly limited in the choice of modification on the benzene ring of the phenoxy group. In addition, synthesis of the compound is rather difficult since the functional group for the labeling inherently has a high reactivity. In contrast, the compound having the functional group for the labeling purpose on the side of the nitrogen atom at 10-position of the acridinium ring has no functional group for the labeling on the side of the leaving group, and therefore, modification of the leaving group is not so difficult.
European Patent Application Laid-Open EP 82636A (Patent family: U.S. Pat. No. 4,946,958) proposes an acridinium ester compound wherein the functional group for the labeling is an active derivative of a carboxylic acid which is present as a substituent on the phenoxy group of the leaving group in the ester moiety. In this type of the acridinium ester, the acridinium ring is highly electron attractive and the carbonyl group is susceptible for nucleophilic attack, and the compound is unstable and easily hydrolyzed. The stability of the compound is insufficient for practical use.
European Patent Application Laid-Open EP 324202A (Patent family: U.S. Pat. No. 5,521,103) proposes an acridinium ester compound and an acridinium acylsulfonamide compound wherein carboxymethyl group for the labeling purpose is present on the nitrogen atom at 10-position of the acridinium ring. In the compounds of the European Patent Application Laid-Open EP 324202A, the carboxymethyl group on the nitrogen at 10-position is a rather large sized substituent, and steric repulsion is generated between the hydrogen atoms on the carbon atoms at 4- and 5-positions of the acridinium ring and the carboxymethyl group. It is estimated that the planar structure of the acridinium ring is no longer retained, and ester bond of such compound is more stable compared to the acridinium compounds retaining its planar structure wherein methyl group is present on the nitrogen atom at 10-position. Such stability is advantageous for use of the compound in the specific binding assay wherein the specific binding reaction such as hybridization of oligonucleotide or an immunoreaction is utilized for the assay.
European Patent Application Laid-Open EP 263657A (Patent family: U.S. Pat. No. 4,745,181) proposes an acridinium ester compound wherein the phenoxy group of the leaving group in the ester moiety has the functional group for the labeling purpose, and 2 substituents are introduced at ortho position of the phenoxy group in the ester moiety for the purpose of improving the stability of the acridinium compound in the solution.
With regard to improvement in the stability of the acridinium compound in the solution, European Patent Application Laid-Open EP 322926A (Patent family: U.S. Pat. No. 5,281,712) proposes an acridinium ester compound wherein 2 electron donor groups are introduced at ortho position and an electron attraction group is introduced at meta or para position of the phenoxy group in the ester moiety; and Japanese Patent Application Laid-Open No. 6(1994)-9566 proposes an acridinium ester compound wherein substituents are introduced at para position of the acridinium ring and at ortho position of the phenoxy group in the ester moiety.
European Patent Application Laid-Open EP 609885A (Patent family: U.S. Pat. No. 5,438,139) proposes an acridinium ester compound of higher stability and higher luminescence efficiency having the functional group for the labeling on the side of the nitrogen atom at 10-position of the acridinium ring. In this compound, carboxymethyl group is present on the nitrogen atom at 10-position of the acridinium ring, and in addition, at least one substituent is present at ortho position of the phenoxy group.
European Patent Application Laid-Open EP 257541A and, EP 273115A (Patent family: U.S. Pat. No. 5,468,646) propose an acridinium acylsulfonamide compounds which are estimated to be more stable than the acridinium ester compound.
European Patent Application Laid-Open EP 330050A, proposes an acridinium compound wherein solubility in water of the compound is improved by incorporating a hetero atom in the molecule.
European Patent Application Laid-Open EP 353971A (Patent family: U.S. Pat. No. 5,227,489) proposes an acridinium ester compound wherein an ionizable group is incorporated at para position of the phenoxy group to improve hydrophilicity, and hence, stability in liposomes; and Japanese Patent Application Laid-Open No. 5(1993)-255264 proposes an acridinium ester compound wherein sulfonium ion is introduced in the molecule to improve solubility of the compound in water.
Japanese Patent Application Laid-Open No. 5(1993)-255263 proposes an acridinium compound which has a structure different from either of the acridinium phenylester compounds and the acylsulfonamide compounds. In this compound, hydrazino group is present in the moiety of the leaving group to reduce blank value and enable use in the assay of high sensitivity.
Japanese Patent Application Laid-Open No. 6(1994)-158039 proposes an acridinium ester compound wherein 2 acridinium rings are present in one molecule.
In spite of the proposals as described above, the compound of these proposals suffered from various insufficiencies.
The acridinium ester of European Patent Application Laid-Open EP 82636A was unstable when stored in the state of solution, and use of this compound for the chemiluminescent label in a specific binding assay was not practical.
In the acridinium esters of European Patent Application Laid-Open EP 263657A and European Patent Application Laid-Open EP 322926A wherein improvement in stability has been attempted, the functional group having the binding activity with the proteins was present on the part of the phenoxy group in the leaving group, and modification of the phenoxy group had been greatly limited. Synthesis of such compounds were also difficult since the functional group with the binding activity inherently had a high reactivity.
The acridinium acylsulfonamide compounds described in European Patent Application Laid-Open EP 257541A, EP 273115A and European Patent Application Laid-Open EP 330050A also had the functional group involved in the binding on the part of the leaving group, and suffered from structural limitation and difficulty in the synthesis as in the case of the above-mentioned acridinium ester compounds. In addition, these acridinium acylsulfonamide compounds were inferior to the acridinium ester compounds in luminescence efficiency.
The acridinium compounds described in Japanese Patent Application Laid-Open Nos. 5(1993)-255263, 5(1993)-255264, 6(1994)-9566 and 6(1994)-158039 also had the functional group involved in the binding on the part of the leaving group, and suffered from the above-described structural limitation and difficulty in the synthesis.
The acridinium compounds of European Patent Application Laid-Open EP 353971A was designed for use with liposomes, and the compounds could not be used for the chemiluminescent label unless additionally processed.
In contrast to the acridinium compounds as described above, the acridinium compounds described in European Patent Application Laid-Open EP 324202A and EP 609885A had the functional group (binding group) for the labeling provided with an activity to bind to proteins and the like on the part of the nitrogen atom at 10-position of the acridinium ring, and the presence of such functional group also resulted in improved stability. In these compounds, modification on the part of the leaving group was also considerably easy owing to the absence of the functional group for the labeling purpose on the part of the leaving group. Furthermore, synthesis of these compounds were quite convenient since introduction of the functional group for the labeling purpose on the nitrogen atom at 10-position of the acridinium ring could be effected at the final stage of the synthesis. These compounds, therefore, were quite useful.
Furthermore, in the acridinium ester described in European Patent Application Laid-Open EP 609885A stability of the ester bond and luminescence efficiency of the acridinium are improved by the presence of the substituent on the phenoxy group of the leaving group in the ester moiety. Further increase in the luminescence efficiency is desired to enable the specific binding assay of higher sensitivity.